EP2877488B1 - Nouveaux domaines de liaison d'acide nucléique spécifiques à la base modulaires à partir de protéines burkholderia rhizoxinica - Google Patents

Nouveaux domaines de liaison d'acide nucléique spécifiques à la base modulaires à partir de protéines burkholderia rhizoxinica Download PDF

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EP2877488B1
EP2877488B1 EP13776595.4A EP13776595A EP2877488B1 EP 2877488 B1 EP2877488 B1 EP 2877488B1 EP 13776595 A EP13776595 A EP 13776595A EP 2877488 B1 EP2877488 B1 EP 2877488B1
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burrh
dna
sequence
nucleic acid
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EP2877488A2 (fr
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Claudia BERTONATI
Philippe Duchateau
Alexandre Juillerat
George SILVA
Julien Valton
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Cellectis SA
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present invention concerns the field of genetic engineering and the reprogramming of cells functions using protein fusions involving new modular specific nucleic acid binding domains.
  • Fusion proteins of these new engineered binding domains with catalytic domains of different nucleic acid processing enzymes, in particular catalytic domains having endonuclease activity, permit the processing of genomes at desired targeted loci.
  • TALE Transcription Activator Like Effectors
  • RVDs repeat variable dipeptides
  • TALE proteins has so far been described as containing: (i) an N-terminal domain including a translocation signal, (ii) a central DNA-binding domain, and (iii) a C-terminal domain including a nuclear localization signal (NLS) and an acidic activation domain (AD).
  • NLS nuclear localization signal
  • AD acidic activation domain
  • a representative member of this family is AvrBs3 from Xanthomonas vesicatoria (SWISSPROT P14727) that has a 1164 amino acid sequence comprising a N-terminal domain of 288 amino acids (position 1 to 288), a central domain of 593 amino acids (positions 289 to 881), and a C-terminal domain of 283 amino acids (positions 882 to 1164) comprising a NLS and AD (transcription activation domain).
  • the DNA-binding domain which determines the target specificity of each TALE consists of a variable number (generally 12 to 27) of tandem, nearly identical, 33-35 amino acid repeats, followed by a single truncated repeat.
  • AvrBs3 DNA-binding domain (SEQ ID NO. 1) comprises 17 repeats of 34 amino acids and a truncated repeat of 15 amino acids.
  • the "repeat-variable di-residue” (RVD), which represents the variable residues in the repeat determines the specificity of interaction with the nucleotide base of the DNA target, in a code-like fashion with some degeneracy.
  • RVD refers the specificity of interaction with the nucleotide base of the DNA target, in a code-like fashion with some degeneracy.
  • the four most common RVDs are HD with respect to c, NI with respect to a, NG with respect to t and NN with respect to g ((Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Bogdanove and Voytas 2011), WO 2011/072246 ).
  • TAL effectors have been, for instance fused to a nuclease catalytic head to form specific nucleases (TALE-Nuclease) creating thereby new tools, especially for genome engineering applications, that have proven efficiency in cell-based assays in yeast, mammalian cells and plants (Cermak, Doyle et al. 2010; Christian, Cermak et al. 2010; Geissler, Scholze et al. 2011; Huang, Xiao et al. 2011; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011; Morbitzer, Elsaesser et al. 2011; Mussolino, Morbitzer et al.
  • a first level of difficulty occurs at the polynucleotide level to clone the repeat sequences due to the fact that restriction sites and PCR primers are basically the same for each repeat. Under these conditions, it gets difficult to perform routine lab procedures to check that the repeats have been cloned properly, in the good number and in the right order. This is although essential to achieve proper expression of a DNA binding protein that is expected to show specificity with a desired nucleic acid sequence.
  • a second level of difficulty occurs when the polynucleotide sequences are included in vectors for heterologous expression, in particular when using viral vectors.
  • DNA tandem repeat motifs from TALE scaffold are generally incompatible with lentiviral vector system due to some internal sequence recombinations. This particularly limits the current use of TALE proteins into primary cells, which are generally not permissive towards classical gene transfer technologies.
  • TALE derived proteins have also been reported in certain cell types, like for instance in mice, or in relation with epigenetic modifications, so that alternative or complementary solutions to improve TALE derived protein are still actively sought.
  • the present inventors have identified putative proteins from the bacterial endosymbiont Burkholderia rhizoxinica and others from a marine organism, displaying highly polymorphic modules having specific DNA binding activity, while having very different sequence (less than 40 % identity) in comparison with TALE repeats. These proteins have also completely different N and C terminal domains.
  • the modules found in these proteins have higher sequence variability than TALE repeats and can although be assembled to engineer new base per base specific binding domains (MBBBD) to target nucleic acid sequences in genomes. These modules confer better sequence stability when they are assembled and expressed in living cells as nucleic acid binding domains.
  • the present invention concerns new modular base-per-base specific nucleic acid binding domains (MBBBD) derived from a newly identified protein from the bacterial endosymbiont Burkholderia Rhizoxinica, namely EAV36_BURRH.
  • EAV36_BURRH a newly identified protein from the bacterial endosymbiont Burkholderia Rhizoxinica
  • Other newly identified proteins from the bacterial endosymbiont Burkholderia Rhizoxinica are described herein, namely E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins and from other similar proteins identified from marine organisms metagenomic database referred to as JCVI_A and JCVI_B and ECR81667.
  • MBBBD modular base-per-base binding domains
  • a Parallel may be made with the repeat domains of TALE proteins from Xanthomonas.
  • the modules in these binding domains display less than 40 % sequence identity with TALE common repeats and much more sequence variability.
  • most modules from these proteins display amino acid variability only in position 13, and not in position 12, whereas variability is observed both in positions 12 and 13 in the variable di-residues (RVDs) of TALE proteins.
  • RVDs variable di-residues
  • base specificity may rely only on position 13 of the modules by merely following a one base/one amino acid code.
  • These proteins display also different N and C-terminal domains, which are much shorter than in TALE proteins.
  • the different domains from said proteins are useful to engineer new proteins or scaffolds having binding properties to specific nucleic acid sequences. Assembling the different modules into new MBBBDs allows targeting almost any nucleic acid sequence in a genome.
  • the MBBBDs can thereby be fused to different catalytic domains to process DNA at the locus of a target nucleic acid sequence, especially nuclease and transcriptional activators.
  • the invention pertains to new rare-cutting endonucleases derived from these polypeptides, with improved specificity or cleavage activity towards a specific locus. Chimeric proteins resulting from the assembly of the different domains from said new modular proteins with functional domains of TALE-like proteins are also disclosed herein.
  • the inventors have conceived different fusion or hybrid proteins deriving from the above polypeptides and polynucleotides and methods to use same.
  • the invention pertains to E5AV36_BURRH modules assembled to form modular base-per-base binding domains (MBBBD).
  • modular base-per-base binding domains is meant a succession of polypeptide modules assembled in order to respectively target a nucleic acid base in a given nucleic acid target sequence.
  • Such MBBBD can be fused to catalytic domains in order to process DNA at a locus defined by a nucleic acid target sequence, especially to a transcription activator, such as VP16 or VP64 or to some repression factors such as for example KRAB (kruppel-associated box) domain.
  • a transcription activator such as VP16 or VP64
  • KRAB kruppel-associated box
  • the MBBBD of the invention are fused to a nuclease catalytic head, especially catalytic domains from Fok-I, to form specific endonucleases, which allow dimerization of Fok-1.
  • the MBBBDs have several advantages over TALE-repeats.
  • the fact that the modules can display non repeated sequences provides the MBBBDs with improved modularity.
  • MBBBD are likely to be processed more easily using PCR, cloning methods and viral delivery methods because polynucleotide sequences encoding the modules are not identical to each other.
  • MBBBDs allow fusions with further nuclease domains such as I-Tevl, making them active under monomeric form as well.
  • the resulting fusion proteins therefore form a new class of engineered endonucleases useful for gene targeting and edition of genomes.
  • Hybrid TALE-like proteins can be also created by combining polypeptide domains (modules, N or C terminals) from the above E5AV36 protein with those of currently existing, natural or engineered TALEs of AvrBs3-like proteins.
  • polypeptide domains modules, N or C terminals
  • Such new chimeric TALE-like proteins can be assembled using the methods already well-established in the art for engineering TALE domains, in particular by sub-cloning the sequences encoding modules or repeats in polynucleotide vectors, for instance, by using Golden Gate cloning method.
  • the protein domains from E5AV36 (module domain, N-terminal domain, C-terminal domain) will be used in combination with the complementary domains of classical TAL effectors.
  • Fusions of catalytic domains to the BURRH polypeptides according to the invention may be N-terminal or C-terminal fusions, with any appropriate linkers or truncations.
  • E5AV36_BURRH modules can also be used as template to build new artificial repeats for TALE-like proteins.
  • Such artificial repeat arrays can be created by introducing mutations into their sequences or by introducing new RVDs into repeats or modules. Key positions at the N/C-terminal domains of the protein can be partially or totally degenerated to modulate DNA affinity as well as interactions with other cofactors. An extensive screening may be also carried out to identify new modules and new RVD-like structures throughout the genomes diversity.
  • the present inventors Upon an extensive search for proteins that may display DNA binding properties throughout a selection of genomes, the present inventors have unexpectedly identified 4 proteins from the microorganism Burkholderia rhizoxinica displaying a modular structure. These modules share a low identity with TALE proteins and have completely different N and C-terminals. Interestingly, the modules of these proteins display more variability than AvrBs3-like repeats and their amino acids in position 12 and 13 significantly differ from those at play in Xanthomonas.
  • Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight.
  • the endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. Its 3.75 Mb genome, which consists of a chromosome and two strain-specific plasmids, was recently sequenced by Lackner, Moebius et al. 2011. Unlike TALE proteins, the DNA binding protein derived from Burkholderia rhizoxinica do not display a transactivator domain and very few is known about the biology of this microorganism.
  • the present disclosure relates to the discovery and identification of new modular proteins obtainable from the different domains of these four proteins:
  • EAV36_BURRH, E5AW43_BURRH and E5AW45_BURRH proteins are flanked by short C and N terminal domains, which do not appear to contain either an acidic domain or a NLS.
  • EAV36_BURRH appears to contain 20 modules and a shorter N- and C-termini.
  • E5AW45_BURRH numbers 27 modules and has N- and C-termini very similar to EAV36_BURRH.
  • E5AW43_BURRH and E5AW46_BURRH are much shorter polypeptides.
  • E5AW43_BURRH has only 6 modules, whereas E5AW46_BURRH does not appear to have any.
  • the N- and C-termini of E5AW43_BURRH and E5AW46_BURRH are very similar to EAV36_BURRH.
  • EAV36_BURRH, E5AW43_BURRH and E5AW45_BURRH proteins are currently annotated in Cog database [http://www.ncbi.nlm.nih.gov/COG] as being: "AraC-type DNA-binding domain-containing proteins".
  • the disclosure relates to the use of these proteins, and more generally of AraC-type DNA binding domains, and more especially modules thereof, for engineering fusion proteins having modular base per base sequence specific binding domains.
  • DIALIGN 2 improvement of the segment-to-segment approach to multiple sequence alignment. Bioinformatics 15, 211 - 218 ).
  • amino acids X 1 and X 2 found in positions 12 and 13 of these modules are more particularly: NI, ND, NG, NA, **, NT, NS, NR, NK, KG and N* (where * means that a deletion appears in the alignment made of the different module sequences as shown in Table 27).
  • Position 12 is mainly represented by N, whereas position 13 is more variable, which suggests that the specificity with respect to nucleobases could rely more particularly on position 13.
  • NT, **, KG, and NR appear to be additional di-residues not occurring in Xanthomonas TALE proteins.
  • nucleotide base specificity could even be determined only by X 2 (position 13) of each module, position 12 (X 1 ) being preferably N, thereby defining a one amino-acid/base code recognition. This would form the first code ever linking one amino acid to one base for specific recognition.
  • This code appears to be primarily based on the following correspondences (AA: amino acid preferably in position 13 of the module): Primary code AA Nucleotide base I A G T D C N G
  • the symbol "*" star means a gap i.e. that there is no position aligned with position 13 using clustal alignment of the different modules.
  • modules from the above proteins also comprise less than 33 amino acids.
  • the matrix in Table 28 details the percentages of identity found between each of the different modules of the BURRH proteins and the following representative AvrBs3 repeat sequence: AvrBs3 LTPEQVVAIASXXGGGKQALETVQRLLPVLCQAHG (SEQ ID NO.10)
  • the percentages of sequence identities for the different modules with respect to the above AvrBs3 repeat are indicated in bold in this matrix.
  • the identity is comprised between 23 % (E5AV36_2) and 47 % (E5AW45_24 and E5AW45_27).
  • Polynucleotide sequences encoding the BURRH proteins E5AV36, E5AW43, E5AW45 and E5AW46 are also described herein. They are respectively referred to as SEQ ID NO.113 (E5AV36), SEQ ID NO.114 (E5AW43), SEQ ID NO.112 (E5AW45) and SEQ ID NO.115 (E5AW46).
  • This search has permitted to identify the following polynucleotide sequence of so far unreported function encoded by genomic DNA isolated from marine organism sample.
  • JCVI_ORF_1096675837214 SEQ ID NO.116
  • JCVI_ORF_1096688227496 SEQ ID NO.117
  • JCVI_ORF_1096688227494 SEQ ID NO.118
  • JCVI_ORF_1096675837216 SEQ ID NO.119
  • JCVI_ORF_1096688327480 SEQ ID NO.120
  • N-terminal and C-terminal of these proteins have been aligned with those from the BURRH proteins: • Aligned N-ter sequences of the following sequences are presented in Table 32: 1) JCVI_B N-ter (SEQ ID NO.75) 76 AA 2) JCVI_A N-ter (SEQ ID NO.74) 66 AA 3) AvrBs3 N-ter (SEQ ID NO.6) 287 AA 4) E5AV36_BURRH N-ter (SEQ ID NO.7) 82 AA 5) E5AW45_BURRH N-ter (SEQ ID NO.9) 83 AA 6) E5AW43_BURRH N-ter (SEQ ID NO.8) 83 AA • Aligned C-ter sequences of the following sequences are presented in Table 33: 1) EAW43_BURRH C-ter (SEQ ID NO.65) 30 AA 2) E5AW45_BURRH C-ter (SEQ ID NO.66) 30 AA 3) E5AV36
  • the module polypeptides of 33 amino acids from the three metagenomic proteins have been aligned using Clustal multiple alignment ( Figure 5 ). These modules also display a higher degree of polymorphism than what can be found among Xanthomonas TALEs. It is although interesting to observe from the logotype and occurrence matrix of Figures 6 and 7 , that a stretch of 10 amino acids, from position 5 to 14, is highly conserved: DIVSIAS X' 1 X' 2 G,
  • Amino acids X' 1 and X' 2 found in positions 12 and 13 of these modules are more particularly: HI, HD, HG, HS, HA, HH, HN, NN, NT and RN.
  • the di-residues HH, HS, NT, HK and RN do not appear to occur in Xanthomonas TALE proteins
  • Position 12 mostly displays H, whereas position 13 is more variable, which suggests that the specificity with respect to the different nucleobases could also rely more particularly on position 13.
  • a primary embodiment of the disclosure is a method to identify putative genomic sequences that may encode modules having specificity to nucleic acid bases.
  • the identification of module sequences according to the invention has come across the following difficulties:
  • the disclosure provides with an approach based on occurrence of repeated structures in putative proteins without taking into account the Xanthomonas TALEs known amino acid sequences.
  • the method is based, as a first screening, on the identification of aminoacidic sequences containing module motifs of variable length (between 20 and 50 aa) using a large variety of computational techniques. Then the candidate sequences are submitted to secondary structure predictions. All the candidates whose module motifs display a high content of alpha helices joined by small loops (whose primary sequence is highly polymorphic) are kept. Finally the entire sequences of the candidates (not only their module motives) are modelled on the available 3D structures. This step allows the identification of the correct number of domains constituting the entire candidate sequences as well as a first functional identification of the key residues regulating the activity of the new putative DNA binding proteins.
  • said method has permitted the identification of proteins referred to as being related to the AraC protein family.
  • proteins of the AraC family have been described as containing DNA-binding domains having the ability of establishing DNA-base contacts (Bustos and Schleif 1993).
  • modular sequences have not been yet reported in connection with AraC DNA binding domains.
  • one aspect of the present disclosure concerns the use of polypeptide sequences referred to in databases as belonging to the AraC protein family as a source of new modules for engineer base per base specific DNA binding domain.
  • DNA binding domains from protein referred to as AraC proteins in genomic databases especially those domains having nucleic acid base specificity, to form fusion proteins for recognition of specific nucleic acid target sequences, is described herein.
  • DNA recognition protein domains may be assembled in order to pair off with a specific nucleic acid base sequence and be fused to catalytic domains to form a new generation of binding proteins.
  • polypeptides derived from the BURRH proteins E5AV36, E5AW43, E5AW45 and E5AW46 and from the metagenomic JCVI_A, JCVI_B and ECR81667 proteins are the polypeptides derived from the BURRH proteins E5AV36, E5AW43, E5AW45 and E5AW46 and from the metagenomic JCVI_A, JCVI_B and ECR81667 proteins. These polypeptides may consist of the whole proteins or of their different domains as previously described especially the different modules, N and C-terminal domains of these proteins.
  • the disclosure encompasses polypeptides variants of the above polypeptides that share at least 70%, preferably at least 80 %, more preferably at least 90 % and even more preferably at least 95 % identity with the sequences provided in this patent application.
  • polypeptides comprising a polypeptide sequence that has at least 60%, preferably 70%, more preferably at least 80%, again more preferably at least 90 %, 95 % 97 % or 99 % sequence identity with any of the above disclosed polypeptide sequences encoding modules, N or C-terminals.
  • the invention relates to the use any polypeptide of sequence SEQ ID NO.11 to 30 as a new or alternative module. Also described herein is the use of any of the polypeptides of sequence SEQ ID NO.7 to 9 or SEQ ID NO. 74 to 76 as new or alternative N-terminal domain, and/or any of said polypeptides of sequence SEQ ID NO.64 to 66 or SEQ ID NO. 109 to 111 as a new or alternative C-terminal, in particular for introduction into existing AvrBs3-like TALE proteins (chimeric proteins).
  • the disclosure also relates to a polypeptide module or modular binding domain of an engineered protein that comprises a module sequence from a protein of the AraC family, especially a module sequence of 30 to 40 amino acids, preferably from 30 to 33 amino acids.
  • polypeptide modules according to the invention are particularly useful to engineer "artificial" nucleic acid binding domains.
  • artificial is meant that they are assembled or modified to bind a desired nucleic acid sequence, said desired target sequence being different from that initially recognized by BURRH protein E5AV36 in the wild.
  • the assembly is generally made by selecting the modules in respect of the affinity of each module to a given nucleic acid base, preferably on a base per base basis.
  • the selection can be made in particular by reference to said one amino-acid/one base code recognition established by the inventors.
  • Said one amino-acid/one base code recognition is based on the following correspondences (AA: amino acid preferably in position 13 of the module): Primary code AA Nucleotide base I A G T D C N G
  • the symbol "*" star means a gap i.e. that there is no position aligned with the amino acid in position 13 using clustal alignment of the different modules.
  • This straightforward code according to the present invention may also be used to modify the specificity of the polypeptide modules by directly introducing mutations in any of the module polypeptides described previously, especially in position 13.
  • the polynucleotide encoding the artificial nucleic acid binding domains of the invention can be assembled by cloning the polynucleotide sequences encoding the different polypeptides by the methods known in the art or by using a solid phase and Type IIS restriction enzymes as described in WO2013/017950 with respect to repeats from TAL binding domains, or even by automated polynucleotide synthesis.
  • the produced polynucleotides can then be cloned into various expression or replication vectors to be transfected into living cells.
  • modules of 32, 31 or less amino acids such as those having identity to SEQ ID NO. 30, 38, 41, 50 and 63 can be used into such artificial nucleic acid binding domains.
  • All the polypeptide modules or mutations according to the present invention can also be introduced into, or assembled with, TAL repeats, to form chimeric MBBBDs (see chimeric proteins).
  • Identity refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting.
  • BLASTP may also be used to identify an amino acid sequence having at least 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence similarity to a reference amino acid sequence using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score.
  • BLASTP "Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other.
  • Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The same applies with respect to polynucleotide sequences using BLASTN.
  • TALE-like polypeptide any polypeptide or protein comprising a binding domain formed by at least two repeats, preferably at least 5, more preferably at least 10, even more preferably at least 14 repeats from a TALE protein having more than 80 % identity with AvrBs3 from Xanthomonas, each of said repeat having specificity for a nucleic acid base.
  • the repeats do not overlap and form a succession of repeats comprising RVDs.
  • RVD sequence may be modified by assembling repeats together to form engineered TALE-like binding domains, thereby allowing targeting any desired sequence in-vivo or in-vitro.
  • modules as disclosed herein may replace some of the AvrBs3-like repeats in such proteins to form new TALE-like chimeric polypeptides.
  • Some modules from the polypeptides according to the disclosure comprise variable residues in position 12 and 13, in particular NT, **, KG, NR, RN, HS, HH and/or HK which may be independently introduced in any existing TALE repeats or in any TALE-like polypeptide as described herein, to improve or modulate their specificity with respect to their cognate nucleic acid bases.
  • polypeptides according to the disclosure previously described may be fused with any other polypeptides to form single chain, monomer or multimer proteins.
  • the above polypeptides can be fused with catalytic domains in order to activate or inactivate transcription or translation activity or process genetic material, within or adjacent to the nucleic acid sequence targeted by the MBBBD.
  • Said catalytic domain can have cleavage activity, either a cleavase activity either a nickase activity, more broadly a nuclease activity but also a polymerase activity, a kinase activity, a phosphatase activity, a methylase activity, a topoisomerase activity, an integrase activity, a transposase activity, a ligase, a helicase or recombinase activity as non-limiting examples.
  • the polypeptides are fused to a catalytic domain which has an endonuclease activity in a monomeric or dimeric form.
  • Suitable domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (1997 )) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998 )); the p65 subunit of nuclear factor kappa B ( Bitko & Barik, J. Virol. 72:5610-5618 (1998 ) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997 )); Liu et al., Cancer Gene Ther.
  • HSV VP16 activation domain see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (1997 )
  • nuclear hormone receptors see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373
  • chimeric functional domains such as VP64 ( Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33 ), and degron ( Molinari et al., (1999) EMBO J. 18, 6439-6447 ).
  • Additional exemplary activation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 ( Seipel et al., EMBO J. 11, 4961-4968 (1992 ) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol. Endocrinol.
  • Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP, and TRAB1.
  • OsGAI OsGAI
  • HALF-1 C1, AP1, ARF-5, -6, -7, and -8
  • CPRF1, CPRF4, MYC-RP/GP TRAB1.
  • Exemplary repression domains include, but are not limited to, KRAB A/B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.
  • TIEG TGF-beta-inducible early gene
  • MBD2 MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.
  • Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al. (1996) Plant Cell 8:305-321 ; and Wu et al. (2000) Plant J. 22:19-27 .
  • polypeptides may also be fused with reporter or selection markers such as GFP and GUS as non limiting examples.
  • Said catalytic domain has preferably an enzymatic activity selected from the group consisting of nuclease activity, polymerase activity, kinase activity, phosphatase activity, methylase activity, topoisomerase activity, integrase activity, transposase activity or ligase activity.
  • the catalytic domain fused to the MBBBD polypeptides of the present disclosure can be a transcription activator or repressor (i.e. a transcription regulator), or a protein that interacts with or modifies other proteins such as histones.
  • Non-limiting examples of nucleic acid processing activities of said fusion MBBBD polypeptides of the present disclosure include, for example, creating or modifying epigenetic regulatory elements, making site-specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.
  • Catalytic domains that may be fused to the MBBBD polypeptides can be selected, for instance, from the group consisting of proteins MmeI, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-Tev-I, I-TevII, I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB, Endodeoxyribonucle
  • amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the peptide. Such variant comprise, for example, deletions from, or insertions or substitutions of residues within the amino acid sequence. Any combination of deletion, insertion or substitutions may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • the catalytic domain is preferably a nuclease domain and more preferably a domain having nuclease activity, like for instance I-Tev-I, Col E7, NucA and Fok-I.
  • said polypeptides that specifically target nucleic acid sequence of interest may be fused to any catalytic domains that require dimerization for activity.
  • said polypeptide may be fused to the type IIS FokI endonuclease domain or functional variant thereof which functions independently of the DNA binding domain and induces nucleic acid double-stranded cleavage as a dimer (Li, Wu et al. 1992; Kim, Cha et al. 1996).
  • Amino acid sequence of Fokl variants can be prepared by mutations in the DNA, which encodes the catalytic domain. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence.
  • Said nuclease domain of Fokl variant according to the present invention comprises a fragment of a protein sequence having at least 80%, more preferably 90%, again more preferably 95 % amino acid sequence identity with the protein sequence of Fokl (SEQ ID NO.123).
  • the targeted nucleic acid sequence of interest are preferably selected with respect to each other, such that the binding of the two fusion polypeptides to their respective target sites places each monomers of the endonuclease in a spatial orientation that allows the formation of a functional cleavage domain by dimerizing.
  • the spacer of the targeted nucleic acid sequences can be selected or varied to modulate MBBD nuclease specificity and activity.
  • the near edge of the target sites are separated by 5 to 50 nucleotides, preferably by 10-30 nucleotides or 25-40 nucleotides.
  • said fusion protein is a monomeric MBBBD-nuclease.
  • a monomeric MBBBD-nuclease is a MBBBD that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered MBBBD modules with the catalytic domain of I-TevI.
  • I-Tevl catalytic domain corresponds to the protein domain or module of an enzyme containing the active site of said enzyme; by active site is intended the part of said enzyme at which catalysis of the substrate occurs.
  • I-Tevl catalytic domain can provide nuclease activity.
  • nuclease catalytic domain is intended the protein domain comprising the active site of an endonuclease enzyme. Such nuclease catalytic domain may generate a cleavage in a nucleic acid target sequence that corresponds to either Double Strand Break (DSB) (cleavase activity) in a nucleic acid target or a single strand break in a nucleic acid target sequence (nickase activity).
  • DSB Double Strand Break
  • nickase activity nickase activity
  • Said catalytic domain can be I-Tevl or a variant thereof.
  • said catalytic domain is a variant of catalytic domain of I-Tevl designed from the N-terminal region of I-Tevl.
  • Said catalytic domain comprises a part of the protein sequence SEQ ID NO. 413.
  • said I-Tevl catalytic domain corresponds to the amino acid sequence of SEQ ID NO. 416 or SEQ ID NO: 417.
  • amino acid sequence variants of the catalytic domain I-Tevl can be prepared by mutations in the DNA, which encodes the catalytic domain.
  • Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • said catalytic domain of I-Tevl comprises a fragment of a protein sequence having at least 80%, more preferably 90%, again more preferably 95 % amino acid sequence identity with the protein sequence SEQ ID NO. 413.
  • said catalytic domain of I-Tevl comprises a protein sequence having at least 80%, more preferably 90%, again more preferably 95% amino acid sequence identity with the protein sequence SEQ ID NO. 416 or SEQ ID NO. 417.
  • Tevl fused MBBBD nuclease interacts with two regions in target nucleic acid sequence: the recognition site and the cleavage site.
  • Optimal distances in the target nucleic acid sequence for the relative positioning of the binding and cleavage modules in the Tevl fused MBBBD polypeptide have been determined.
  • the present invention relates to a MBBBD polypeptide capable of targeting a nucleic acid sequence that comprises a recognition site spaced away from said I-Tevl cleavage site by an optimal distance to increase DNA processing activity.
  • Increased DNA processing activity refers to an increase in the detected level of MBBBD nuclease processing activity against a target nucleic acid sequence.
  • nucleic acid processing activity refers to a cleavage, either a cleavase activity or a nickase activity.
  • optimal distance is intended the distance between said recognition site and I-Tevl cleavage site allowing an increase in DNA processing activity of the Tevl chimeric endonuclease.
  • An optimal distance is considered when it provides at least a 5% increase efficiency of DNA processing activity, more preferably 10%, again more preferably 15%, again more preferably 20%, again more preferably 25%, again more preferably 50%, again more preferably greater than 50%.
  • DNA binding recognition site is also chosen based upon its optimal spacer between the residue preceded the first nucleic acid base of DNA binding recognition site and the terminal G base of the I-Tevl cleavage site.
  • the optimal spacer distance is between 1 to 50 bp, more preferably between 4 to 12 bp, again more preferably is 4, 5, 6, 7, 8, 9, 10, 11 or 12 bp.
  • the nuclease is a meganuclease (homing endonuclease) or variant thereof.
  • Naturally-occurring meganucleases recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family.
  • Exemplary homing endonucleases include I-Sce I, I-Chu I, I-Cre I, I-Csm I, PI-Sce I, PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu
  • the homing endonuclease according to the invention is a LAGLIDADG endonuclease such as I-SceI, I-CreI, I-CeuI, I-OnuI, I-MsoI, and I-DmoI.
  • said LAGLIDADG endonuclease is I-CreI. Wild-type I-CreI is a homodimeric homing endonuclease that is capable of cleaving a 22 to 24 bp double-stranded target sequence.
  • homing endonuclease variants such as I-CreI may be homodimers (meganuclease comprising two identical monomers) or heterodimers (meganuclease comprising two non-identical monomers). It is understood that the scope of the present invention also encompasses the homing endonuclease variants per se, including heterodimers ( WO2006097854 ), obligate heterodimers ( WO2008093249 ) and single chain meganucleases ( WO03078619 and WO2009095793 ) as non limiting examples, able to cleave one of the sequence targets in the cell genome. The invention also encompasses hybrid variant per se composed of two monomers from different origins ( WO03078619 ).
  • the invention encompasses both wild-type and variant endonucleases.
  • the endonuclease according to the invention is a "variant" endonuclease, i.e. an endonuclease that does not naturally exist in nature and that is obtained by genetic engineering or by random mutagenesis.
  • the variant endonuclease according to the invention can for example be obtained by substitution of at least one residue in the amino acid sequence of a wild-type, endonuclease with a different amino acid. Said substitution(s) can for example be introduced by site-directed mutagenesis and/or by random mutagenesis. In the frame of the present invention, such variant endonucleases remain functional, i.e.
  • variant endonuclease according to the invention cleaves a target sequence that is different from the target sequence of the corresponding wild-type endonuclease. Methods for obtaining such variant endonucleases with novel specificities are well-known in the art.
  • Said catalytic domain might be at the N-terminal part or C-terminal part of said MBBBD.
  • Said catalytic domain is fused to MBBBD by a peptide linker.
  • Peptide linker acts as a communication device between the MBBBD polypeptide and catalytic domain to act in concert for nucleic acid cleavage.
  • Said peptide linkers can be of various sizes, preferably from 2 to 50 amino acids, more preferably from 3 to 10 amino acids and can be selected from the group consisting of NFS1, NFS2, CFS1, RM2, BQY, QGPSG, LGPDGRKA, 1a8h_1, 1dnpA_1, 1d8cA_2, 1ckqA_3, 1sbp_1, 1ev7A_1, 1alo_3, 1amf_1, 1adjA_3, 1fcdC_1, 1al3_2, 1g3p_1, 1acc_3, 1ahjB_1, 1acc_1, 1af7_1, 1heiA_1, 1bia_2, 1igtB_1, 1nfkA_1, 1au7A_1, 1bpoB_1, 1b0pA_2, 1c05A_2, 1gcb_1, 1bt3A_1, 1b3oB_2, 16vpA_6, 1dhx_1, 1b8aA_1and 1qu6
  • the peptide linker that can link said catalytic domain to the MBBBD polypeptide according to the method of the present invention can be selected from the group consisting of GRSGSDP (SEQ ID NO: 489), QGPSG (SEQ ID NO: 487), IA (SEQ ID NO.90) or SG (SEQ ID NO: 491).
  • Peptide linkers between the MBBBD polypeptide and the catalytic domain can be constructed to be either flexible or positionally constrained to allow for the most efficient activity targeted nucleic acid processing.
  • Example 1 shows that the above polypeptides have the ability to dimerize when fused to the catalytic domain of the nuclease Fok-I.
  • a fusion of BurrH_36 with Fok-I has been achieved to form a sequence specific nuclease being able to cut a putative artificial nucleic acid target.
  • this fusion experiment revealed that, contrary to TALE-Nucleases, there was no requirement for T in the target DNA sequence for the first module to bind said nucleic acid target. It is unclear at the moment whether it is due to the N-terminus (SEQ ID NO.7) or to the first module (SEQ ID NO.11) of the BurrH protein.
  • these polypeptides provide a significant advantage over the TALE-Nuclease of the prior art in this regard.
  • the invention also provides modular polypeptides or N-terminal sequences to alleviate the requirement of a T in sequences to be targeted by a TALE or TALE-like binding domain.
  • Such module or N-terminal domain according to the invention may thus be introduced in TALE or TALE-like repeat binding domains to overcome the requisite T nucleotide at position -1 in nucleic acid target sequences.
  • Truncations, spacers and linkers may be added by one skilled in the art to the polypeptides according to the invention to optimize their binding activity or the catalytic activity conferred by their catalytic domains.
  • the catalytic domain that is capable of processing genetic material withinin or adjacent the nucleic acid target sequence of interest can be fused to the N- or C-terminus part of said binding domains of the invention.
  • two catalytic domains having complementary or distinct activities are fused to both N-terminus and C-terminus parts of said binding domains.
  • polypeptides and fusion proteins previously described can be used to create chimeric proteins, which incorporate sequences from AvrBs3-like proteins, in particular repeats, N-terminal or C-terminal sequences thereof.
  • the disclosure provides engineered TALE-like proteins with a binding domain comprising a mix of the modules according to the invention and of AvrBs3-like repeats.
  • new scaffolds can be derived from AvrBs3-like proteins comprising a module, N or C terminals, or any functional part of the polypeptides from E5AV36, E5AW43, E5AW45, E5AW46, JCVI_A, JCVI_B and ECR81667 previously described.
  • the chimeric protein of the present invention can be derived from any naturally occurring TAL effectors, such as those described by (Moscou and Bogdanove 2009) and in WO 2011072246 ., that comprise repeats of 33 to 35 amino acids, wherein two critical amino acids located at positions 12 and 13 (RVD) mediate specific nucleic acid base recognition.
  • RVDs can be used: HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A.
  • RVDs associated with recognition of the nucleotides C, T, A, G/A and G respectively are selected from the group consisting of NN or NK for recognizing G, HD for recognizing C, NG for recognizing T and NI for recognizing A, TL for recognizing A, VT for recognizing A or G and SW for recognizing A.
  • RVDs associated with recognition of the nucleotide C are selected from the group consisting of N*
  • RVDS associated with recognition of the nucleotide T are selected from the group consisting of N* and H*, where * denotes a gap in the repeat sequence that corresponds to a lack of amino acid residue at the second position of the RVD.
  • critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity.
  • other amino acid residues is intended any of the twenty natural amino acid residues or unnatural amino acids derivatives. All these RVDs can be used in addition to those with respect to the present invention, especially: NT, **, KG, NR, RN, HS, HH and/or HK.
  • chimeric MBBBD protein may be created by combining modules domains from E5AV36, E5AW43, E5AW45, EAW46, JCVI_A, JCVI_B and ECR81667 proteins with repeat domain of AvrBs3-like proteins, by combining modules domains from E5AV36, E5AW43, E5AW45, EAW46, JCVI_A, JCVI_B and ECR81667 proteins with the N- and C-terminal domains of AvrBs3-like proteins, by combining N and C-terminal domains of E5AV36, E5AW43, E5AW45, EAW46, JCVI_A, JCVI_B and ECR81667 proteins with repeat domain of AvrBs3-like proteins, by combining the N-terminal domain of AvrBs3-like proteins with modules domain and C-terminal from E5AV36, E5AW43, E5AW45, EAW46, JCVI_A, JCVI_
  • E5AV36, E5AW43, E5AW45, EAW46, JCVI_A, JCVI_B and ECR81667 with part of N-terminal domain of AvrBs3-like protein, or by combining part of DNA binding modules of E5AV36, E5AW43, E5AW45, EAW46, JCVI_A, JCVI_B and ECR81667 with part of repeat domain of AvrBs3-like protein
  • the protein domains from the E5AV36, E5AW43, E5AW45, E5AW46, JCVI_A, JCVI_B and ECR81667 proteins may be used in combination with the complementary domains of classical TAL effectors.
  • a most preferred chimeric protein comprises modules from E5AV36 with a N-terminal from AvrBs3 (see example 12,
  • the disclosure also concerns the polynucleotides, in particular DNA or RNA encoding the polypeptides and proteins previously described. These polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in prokaryotic or eukaryotic cells.
  • the polynucleotides of SEQ ID NO.112 to 120 correspond to the sequences that have been identified according to the invention in the genomic databases.
  • Polynucleotides according to the disclosure encompass polynucleotides having at least 80 %, preferably at least 90 %, more preferably at least 95 and even more preferably 99 % identity with the above polynucleotide sequences.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a “vector” in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids.
  • Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.
  • orthomyxovirus e. g., influenza virus
  • rhabdovirus e. g., rabies and vesicular stomatitis virus
  • paramyxovirus e. g. measles and Sendai
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus ( Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996 ).
  • viral vectors are viral vectors, more particularly lentiviral vectors.
  • viral vector refers to a nucleic acid construct which carries, and within certain embodiments, is capable of directing the expression of a nucleic acid molecule of interest.
  • the lentiviral vector can include at least one transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
  • Such vector constructs can also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
  • LTRs long terminal repeats
  • the recombinant lentiviral vector may also include a signal which directs polyadenylation, selectable markers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • selectable markers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR
  • such vectors typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.
  • influenza virus rhabdovirus
  • paramyxovirus e. g. measles and Sendai
  • positive strand RNA viruses such as picornavirus and alphavirus
  • double-stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox and canarypox).
  • herpesvirus e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
  • poxvirus e. g., vaccinia, fowlpox and canarypox.
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus ( Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996 ).
  • the present invention relates to a viral vector, preferably a lentiviral vector which comprises polynucleotide encoding MBBBD or MBBBD-fusion protein as described above.
  • a viral vector preferably a lentiviral vector which comprises polynucleotide encoding MBBBD or MBBBD-fusion protein as described above.
  • Any of these vectors can comprise one or more polynucleotide encoding MBBBD or MBBBD-fusion proteins.
  • one vector can comprise two sequences encoding two MBBBD monomers which can recognize different adjacent nucleic acid target sequences and the two protein domains function as subdomains that need to interact in order to process the genetic sequence.
  • One vector can also comprise two sequences encoding two monomeric MBBBD which recognize and process two different nucleic acid target sequences.
  • HIV particle refers to a virus which carries at least one gene of interest.
  • the virus may also contain a selectable marker.
  • HIV type 1 (HIV-1) based vector particles may be generated by co-expressing the virion packaging elements and the vector genome in a so-called producer cell, e.g. 293T human embryonic kidney cells. These cells may be transiently transfected with a number of plasmids. Typically from three to four plasmids are employed, but the number may be greater depending upon the degree to which the lentiviral components are broken up into separate units. Generally, one plasmid encodes the core and enzymatic components of the virion, derived from HIV-1.
  • This plasmid is termed the packaging plasmid.
  • Another plasmid encodes the envelope protein(s), most commonly the G protein of vesicular stomatitis virus (VSV G) because of its high stability and broad tropism.
  • This plasmid may be termed the envelope expression plasmid.
  • Yet another plasmid encodes the genome to be transferred to the target cell, that is, the vector itself, and is called the transfer vector.
  • Recombinant viruses with titers of several millions of transducing units per milliliter (TU/ml) can be generated by this technique and variants thereof. After ultracentrifugation concentrated stocks of approximately 10 9 TU/ml can be obtained.
  • the lentivirus is capable of reverse transcribing its genetic material into DNA and incorporating this genetic material into a host cell's DNA upon infection.
  • Lentiviral vector particles may have a lentiviral envelope, a non-lentiviral envelope (e.g., an ampho or VSV-G envelope), or a chimeric envelope.
  • the present disclosure relates to a viral, preferably a lentiviral particle which comprises polynucleotides encoding MBBBD or MBBBD-fusion protein as described above.
  • the present disclosure relates to an in vitro method of processing a nucleic acid target sequence of a cell, comprising: (a) providing a cell containing a target nucleic acid sequence; and (b) introducing into the cell a nucleic acid binding polypeptide such that said polypeptide processes the nucleic acid target sequence.
  • Said nucleic acid binding polypeptide can be designed to recognize any suitable nucleic acid target sequence.
  • processing means that the sequence is considered modified simply by the binding of the polypeptide.
  • Any nucleic acid target sequence can be processed by the present methods.
  • the nucleic acid target sequence can be chromosomal, mitochondrial or chloroplast sequences.
  • a method of processing the genetic material of a cell within or adjacent to a nucleic acid target sequence is provided by introducing into the cell fusion MBBBD polypeptides.
  • Catalytic domain of the fusion protein of the present invention can be a transcription activator or repressor (i.e. a transcription regulator), or a protein that interacts with or modifies other proteins implicated in nucleic acid processing.
  • Non-limiting examples of nucleic acid processing activities of said fusion polypeptides of the present invention include, for example, creating or modifying epigenetic regulatory elements, making site-specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.
  • Said nucleic acid processing activity can refer to a cleavage activity, either a cleavase activity either a nickase activity, more broadly a nuclease activity but also a polymerase activity, a kinase activity, a phosphatase activity, a methylase activity, a topoisomerase activity, an integrase activity, a transposase activity, a ligase, a helicase or recombinase activity as non-limiting examples.
  • cell or cells any prokaryotic or eukaryotic living cells, cell lines derived from these organisms for in vitro cultures, primary cells from animal or plant origin.
  • primary cell or “primary cells” are intended cells taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. These cells thus represent a more valuable model to the in vivo state they refer to.
  • eukaryotic cells refer to a yeast, fungal, plant or animal cell or a cell line derived from the organisms listed below and established for in vitro culture. More preferably, the fungus is of the genus Aspergillus, Penicillium, Acremonium, Trichoderma, Chrysoporium, Mortierella, Kluyveromyces or Pichia.
  • the plant is of the genus Arabidospis, Nicotiana, Solanum, lactuca, Brassica, Glycine, Oryza, Asparagus, Pisum, Medicago, Zea, Hordeum, Secale, Triticum, Capsicum, Cucumis, Cucurbita, Citrullis, Citrus, or Sorghum.
  • the animal cell is of the genus Homo, Rattus, Mus, Sus, Bos, Danio, Canis, Felis, Equus, Salmo, Oncorhynchus, Gallus, Meleagris, Drosophila, or Caenorhabditis;
  • the cell can be a plant cell, a mammalian cell, a fish cell, an insect cell or cell lines derived from these organisms for in vitro cultures or primary cells which have been taken directly from living tissue and established for in vitro culture.
  • cell can be protoplasts obtained from plant organisms listed above.
  • cell lines can be selected from the group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
  • All these cell lines can be modified by the method of the present invention to provide cell line models to produce, express, quantify, detect, study a gene or a protein of interest; these models can also be used to screen biologically active molecules of interest in research and production and various fields such as chemical, biofuels, therapeutics and agronomy as non-limiting examples.
  • Adoptive immunotherapy using genetically engineered T cells is a promising approach for the treatment of malignancies and infectious diseases. Most current approaches rely on gene transfer by random integration of an appropriate T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR). Targeted approach using rare-cutting endonucleases is an efficient and safe alternative method to transfer genes into T cells and generate genetically engineered T cells.
  • TCR T Cell Receptor
  • CAR Chimeric Antigen Receptor
  • the present invention also relates to in vitro methods for use of said polypeptides polynucleotides and proteins previously described for various applications ranging from targeted nucleic acid cleavage to targeted gene regulation.
  • the efficiency of nuclease fusion protein or chimeric protein as referred to in the present patent application e.g. their ability to induce a desired event (Homologous gene targeting, targeted mutagenesis, sequence removal or excision) at a locus, depends on several parameters, including the specific activity of the nuclease, probably the accessibility of the target, and the efficacy and outcome of the repair pathway(s) resulting in the desired event (homologous repair for gene targeting, NHEJ pathways for targeted mutagenesis).
  • the present invention more particularly relates to an in vitro method for modifying the genetic material of a cell within or adjacent to a nucleic acid target sequence.
  • the double strand breaks caused by endonucleases are commonly repaired through non-homologous end joining (NHEJ).
  • NHEJ comprises at least two different processes. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used for the creation of specific gene knockouts.
  • the present invention related to an in vitro method for modifying the genetic material of a cell within or adjacent to a nucleic acid target sequence by using nuclease MBBBD fusion protein according to the invention that allows nucleic acid cleavage that will lead to the loss of genetic information and any NHEJ pathway will produce targeted mutagenesis.
  • the present invention related to an in vitro method for modifying the genetic material of a cell within or adjacent to a nucleic acid target sequence by generating at least one nucleic acid cleavage and a loss of genetic information around said target nucleic acid sequence thus preventing any scarless re-ligation by NHEJ.
  • Said modification may be a deletion of the genetic material, insertion of nucleotides in the genetic material or a combination of both deletion and insertion of nucleotides.
  • homologous is intended a sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 95 % identity, preferably 97 % identity and more preferably 99 %.
  • the present disclosure also relates to a method for modifying target nucleic acid sequence further comprising the step of expressing an additional catalytic domain into a host cell.
  • the present invention relates to a method to increase mutagenesis wherein said additional catalytic domain is a DNA end-processing enzyme.
  • DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, hosphatase, hydrolases and template-independent DNA polymerases.
  • Non limiting examples of such catalytic domain comprise of a protein domain or catalytically active derivate of the protein domain seleced from the group consisting of hExoI (EXO1_HUMAN), Yeast ExoI (EXO1_YEAST), E.coli ExoI, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST).
  • EXO1_HUMAN hExoI
  • EXO1_YEAST Yeast ExoI
  • E.coli ExoI E.coli ExoI
  • Human TREX2 Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST).
  • said additional catalytic domain has a 3'-5'-exonuclease activity, and in a more preferred embodiment, said additional catalytic domain has TREX exonuclease activity, more preferably TREX2 activity ( WO2012058458 ).
  • said catalytic domain is encoded by a single chain TREX polypeptide ( WO2013009525 ). Said additional catalytic domain may be fused to a nuclease fusion protein or chimeric protein according to the disclosure optionally by a peptide linker.
  • the present disclosure relates to a method for inducing homologous gene targeting in the target nucleic acid sequence further comprising providing to the cell an exogeneous nucleic acid comprising at least a sequence homologous to a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the exogeneous nucleic acid.
  • Said exogenous nucleic acid usually comprises a sequence homologous to at least a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the exogenous nucleic acid.
  • said exogenous nucleic acid comprises first and second portions which are homologous to region 5' and 3' of the target nucleic acid, respectively.
  • Said exogenous nucleic acid in these embodiments also comprises a third portion positioned between the first and the second portion which comprises no homology with the regions 5' and 3' of the target nucleic acid sequence.
  • a homologous recombination event is stimulated between the genome containing the target nucleic acid sequence and the exogenous nucleic acid.
  • homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used within said donor matrix. Therefore, the exogenous nucleic acid is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp.
  • shared nucleic acid homologies are located in regions flanking upstream and downstream the site of the break and the nucleic acid sequence to be introduced should be located between the two arms.
  • Said exogenous nucleic acid can comprise a positive selection marker between the two homology arms and eventually a negative selection marker upstream of the first homology arm or downstream of the second homology arm.
  • the marker(s) allow(s) the selection of the cells having inserted the sequence of interest by homologous recombination at the target site.
  • exogenous nucleic acid can be used to knock-out a gene, e.g. when exogenous nucleic acid is located within the open reading frame of said gene, or to introduce new sequences or genes of interest.
  • Sequence insertions by using such exogenous nucleic acid can be used to modify a targeted existing gene, by correction or replacement of said gene (allele swap as a non-limiting example), or to up- or down-regulate the expression of the targeted gene (promoter swap as non-limiting example), said targeted gene correction or replacement.
  • the methods of the disclosure involve introducing a polynucleotide encoding MBBBD polypeptide into a cell.
  • Methods for introducing a polynucleotide construct into bacteria, plants, fungi and animals are known in the art and including as non limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods.
  • Said polynucleotides encoding MBBBD polypeptide may be introduced into a cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomes and the like.
  • transient transformation methods include for example microinjection, electroporation, particle bombardment
  • the MBBD polypeptide may be synthesized in situ in the cell as a result of the introduction of polynucleotide encoding polypeptide into the cell.
  • the MBBBD polypeptide could be produced outside the cell and then introduced thereto.
  • the method for targeting genetic material of a cell comprises providing a cell comprising a nucleic acid target and introducing the polynucleotide encoding MBBBD or MBBBD fusion protein as described above into the cell via a viral particle, and expressing said polynucleotide within the cell.
  • the viral particle comprises the polynucleotide and said polynucleotide is introduced into the cell by contacting said cell with the viral particle under condition that permits infection.
  • Engineered MBBBD polypeptides can be produced by rearranging modules thus allowing the generation of modular domain with novel target nucleic acid specificity.
  • Each different MBBBD modules can be engineered following the recognition code according to the present invention.
  • the present invention relates to a method to produce MBBBD polypeptides capable of binding to any desired nucleic acid target sequence by assembling the different engineered MBBBD modules in the appropriate order.
  • Non-human animals may be generated by introducing MBBBD polypeptide into a cell or a non-human embryo.
  • the present disclosure relates to a method for generating an animal, comprising providing an eukaryotic cell comprising a nucleic acid target sequence into which it is desired to introduce a genetic modification; generating a cleavage within or adjacent to the nucleic acid target sequence by introducing a MBBBD polypeptide according to the present invention; and generating an animal from the cell or progeny thereof, in which cleavage has occurred.
  • the embryo is a fertilized one cell stage embryo.
  • Polynucleotides encoding said MBBBD polypeptides may be introduced into the cell by any of the methods known in the art including micro injection into the nucleus or cytoplasm of the embryo.
  • the method for generating a non-human animal further comprise introducing an exogenous nucleic acid as desired.
  • Said exogenous nucleic acid comprises a sequence homologous to at least a portion of the nucleic acid target sequence, such that homologous recombination occurs between said exogenous nucleic acid and the nucleic acid target sequence in the cell or progeny thereof.
  • the exogenous nucleic acid can include for example a nucleic acid sequence that disrupts a gene after homologous recombination, a nucleic acid sequence that replaces a gene after homologous recombination, a nucleic acid sequence that introduces a mutation into a gene after homologous recombination or a nucleic acid sequence that introduce a regulatory site after homologous recombination.
  • the embryos are then cultures to develop an animal.
  • a non-human an animal in which at least a nucleic acid target sequence of interest has been engineered is provided.
  • an engineered gene may become inactivated such that it is not transcribed or properly translated, or an alternate form of the gene is expressed.
  • the animal may be homozygous or heterozygous for the engineered gene.
  • the present disclosure also related to a method for generating a plant comprising providing a plant cell comprising a nucleic acid target sequence into which it is desired to introduce a genetic modification; generating a cleavage within or adjacent to the nucleic acid target sequence by introducing a MBBD polypeptide according to the present invention; and generating a plant from the cell or progeny thereof, in which cleavage has occurred.
  • Progeny includes descendants of a particular plant or plant line.
  • the method for generating a plant further comprise introducing an exogenous nucleic acid as desired.
  • Said exogenous nucleic acid comprises a sequence homologous to at least a portion of the nucleic acid target sequence, such that homologous recombination occurs between said exogenous nucleic acid and the nucleic acid target sequence in the cell or progeny thereof.
  • Plant cells produced using methods can be grown to generate plants having in their genome a modified nucleic acid target sequence. Seeds from such plants can be used to generate plants having a phenotype such as, for example, an altered growth characteristic, altered appearance, or altered compositions with respect to unmodified plants.
  • polypeptides of the invention are useful to engineer genomes and to reprogram cells, especially iPS cells and ES cells.
  • polypeptides according to the invention can be used as a medicament, especially for modulating, activating or inhibiting gene transcription, at the promoter level or through their catalytic domains.
  • Fusion proteins composed of a binding domain according to the invention and of a catalytic domain with nuclease activity can be used for the treatment of a genetic disease to correct a mutation at a specific locus or to inactivate a gene the expression of which is deleterious.
  • Such proteins can also be used to genetically modify iPS or primary cells, for instance T-cells, in view of injected such cells into a patient for treating a disease or infection.
  • Such cell therapy schemes are more particularly developed for treating cancer, viral infection such as caused by CMV or HIV or self-immune diseases.
  • BurrH_36 (SEQ ID NO.2) was chosen as a starting polypeptide to create new specific nuclease.
  • the two domains were separated by a small DNA sequence containing two BsmBI sites allowing further cloning of the DNA coding for the DNA binding array, a Nuclear Localization Sequence (NLS) and HA tag were added in front of the N-terminal domain and short sequences were added between the different pieces for cloning purpose or to create linkers at the protein level, leading to BurrH_36 scaffold pCLS17028 (SEQ ID NO.121).
  • the DNA coding for the DNA binding array (BurrH_RVD_array1, SEQ ID NO.122) was synthesized according to the human genetic code and cloned in the pUC57 plasmid, by Genecust, leading to pCLS17030.
  • the BurrH_36 scaffold was then sublconed, from pCLS17028, into a yeast expression vector containing a Fokl catalytic head (SEQ ID NO.123) preceded by a short linker sequence, using Ncol and BamHI, leading to pCLS17419 (SEQ ID NO.124).
  • the DNA binding array insert was then subcloned, from pCLS17030 into pCLS17419 using the two BsmBI sites, leading to pCLS17421 (SEQ ID NO.125). All molecular biology steps were done according to standard procedures.
  • yeast target reporter plasmids containing the DNA target collection sequences were constructed as previously described (International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).
  • the BurrH_36 based nucleases were tested at 37°C and 30°C in yeast SSA assay as previously described in WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al.
  • Figure 8 shows the correspondence between the different modules of BurrH_36 with respect to the different DNA targets.
  • Target A is common to the target sequences BURRH_v01, BURRH_v02, BURRH_v03 et BURRH_v04 presented in Table 1
  • Target B is common to the target sequences BURRH_v05, BURRH_v06, BURRH_v07 et BURRH_v08.
  • Target C is common to the target sequences BURRH_v09, BURRH_v10, BURRH_v11 et BURRH_v12.
  • Table 2 Activity of BurrH_36 derived nuclease on pseudo-palindromic sequences targets (two identical recognition sequences are placed facing each other on both DNA strands) in yeast SSA assay at 37°C. "-" indicates that no cleavage activity was detected and +++ a high cleavage activity.
  • Example 2 Nuclease activity of BurrH_36 based constructs in function of target nucleotides in position 0, -1 and -2
  • Nuclease activity of BurrH_36 based nuclease encoded in plasmid pCLS17421 was monitored on a set of 21 targets differing only by their nucleotidic sequences in position 0, -1 and -2 (SEQ ID NO. 139 to 159, see Table 3) as described in Example 1. Similar activities were obtained on all targets (Table 4) indicating an absence of specific sequence requirement for position 0, -1 and -2.
  • Example 3 Nuclease activity of engineered BurrH_36 based constructs containing 18 DNA binding modules
  • the BurrH_36 scaffold in a yeast expression vector, described in example 1 was chosen as receiving scaffold plasmid.
  • the DNA binding array insert was then subcloned, from pCLS18120 (SEQ ID NO. 160) into pCLS17419 using the two BsmBI sites, leading to pCLS18473 (SEQ ID NO. 161). All molecular biology steps were done according to standard procedures.
  • the sequences of the DNA binding array insert are represented in Table 5 below.
  • Table 3 List of all pseudo-palindromic (two identical recognition sequences are placed facing each other on both DNA strands) sequences targets, with various nucleotides in position 0, -1 and -2 used in the yeast SSA assay.
  • BurrH_36 1 FSQSD IVKIA GXXGG AQALQ AVLDL ESMLG KRG 162 BurrH_36 2 FSRDD IAKMA GXXGG AQTLQ AVLDL ESAFR ERG 163 BurrH_36 3 FSQAD IVKIA GXXGG AQALY SVLDV EPTLG KRG 164 BurrH_36 4 FSRAD IVKIA GXXGG AQALH TVLDL EPALG KRG 165 BurrH_36 5 FSRID IVKIA AXXGG AQALH AVLDL GPTLR ECG 166 BurrH_36 6 FSQAT IAKIA GXXGG AQALQ MVLDL GPALG KRG 167 BurrH_36 7 FSQAT IAKIA GXXGG AQALQ TVLDL EPALC ERG 168 BurrH_36 8 FSQAT IAKMA GXXGG AQALQ TVLDL EPALR KRD 169 BurrH
  • the yeast target reporter plasmids containing the DNA target sequences were constructed as previously described (International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).
  • the BurrH_36 based nucleases were tested on the target collection at 37°C in yeast SSA assay as previously described ( WO 2004/067736 and (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al.
  • Targets are designed as pseudo-palindromic sequences (two identical recognition sequences are placed facing each other on both DNA strands).
  • BurrH_36 based nucleases cleavage activity levels on the complete collection of targets in yeast are shown on Table 7.
  • Table 6 List of all pseudo-palindromic (two identical AvBrs3 recognition sequences are placed facing each other on both DNA strands) sequences targets, with various spacer length (ranging from 5 to 40 bp) used in yeast SSA. Minuscule letters represent spacers.
  • DNA Target pCLS18473 cleavage activity Avr05 - Avr06 - Avr07 - Avr08 + Avr09 - Avr10 - Avr11 - Avr12 +++ Avr13 +++ Avr14 +++ Avr15 +++ Avr16 +++ Avr17 +++ Avr18 +++ Avr19 +++ Avr20 +++ Avr21 +++ Avr22 +++ Avr23 +++ Avr24 +++ Avr25 +++ Avr26 +++ Avr27 +++ Avr28 ++ Avr29 ++ Avr30 ++ Avr31 ++ Avr32 ++ Avr33 +++ Avr34 +++ Avr35 +++ Avr36 +++ Avr37 ++ Avr38 + Avr39 + Avr40 +
  • Example 4 Nuclease activity of engineered BurrH_36 based constructs containing 16 DNA binding modules
  • the BurrH_36 scaffold in a yeast expression vector, described in example 1 was chosen as receiving scaffold plasmid.
  • the DNA binding array inserts were then subcloned, from pCLS18123 and pCLS18127 (SEQ ID NO. 218 and SEQ ID NO. 219) into pCLS17419 using the two BsmBI sites, leading to respectively pCLS18476 (SEQ ID NO. 220) and pCLS18480 (SEQ ID NO. 221). All molecular biology steps were done according to standard procedures.
  • the sequences of the DNA binding array insert are represented in Table 8 below.
  • the yeast target reporter plasmids containing the RAGT2.4 and RAGT2.3 DNA target sequences were constructed as previously described (International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).
  • the BurrH_36 based nucleases were tested on their respective target at 37°C in the yeast SSA assay as previously described. Targets are designed as pseudo-palindromic sequences (two identical recognition sequences are placed facing each other on both DNA strands).
  • BurrH_36 based nucleases cleavage activity levels on their respective targets in yeast are shown in Table 10.
  • Table 9 List of the 2 pseudo-palindromic (two identical recognition sequences are placed facing each other on both DNA strands) sequences targets, used in the yeast and mammalian SSA assays. Minuscule letters represent spacers. Name Sequence SEQ ID NO.
  • RAGT2.4 TGTTTATGGTTACTTATatgtgtgtaacaggtATAAGTAACCATAAACA 222 RAGT2.3 TATATTTAAGCACTTATatgtgtgtaacaggtATAAGTGCTTAAATATA 223
  • Table 10 Activity of BurrH_36 derived nuclease on pseudo-palindromic sequences targets listed in Table 9 in yeast SSA assay at 37°C. "- "indicates that no activity was detected and +++ a high activity.
  • DNA Target pCLS18476 cleavage activity
  • pCLS18480 cleavage activity RAGT2.4 +++ - RAGT2.3 - +++
  • Example 5 Nuclease activity of engineered BurrH_36 based constructs containing 16 engineered alternative DNA binding modules
  • the BurrH_36 scaffold in a yeast expression vector, described in example 1 was chosen as receiving scaffold plasmid.
  • the DNA binding array inserts were then subcloned, from pCLS18124 to pCLS18126 (SEQ ID NO. 224 to SEQ ID NO. 226) into pCLS17419 using the two BsmBI sites, leading to pCLS18477 to pCLS18479 respectively (SEQ ID NO. 227 to SEQ ID NO. 229). All molecular biology steps were done according to standard procedures.
  • the sequences of DNA binding array insert of pCLS18477 to pCLS18479 are represented in Tables 11 to 13 respectively.
  • Table 11 Sequences of the 16 module domains of pCLS18477 construct derived from the alignment of the first 5 modules of E5AV36.
  • Module domains pCLS18478 DNA binding array SEQ ID NO. consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG AQALQ AVLDL EPTLR ERG 231 consensus all FSQAD IVKIA GXXGG
  • the yeast target reporter plasmids containing the DNA target sequence (SEQ ID NO. 222, Table 9) were constructed as previously described.
  • the BurrH_36 based nucleases were tested on their respective target at 37°C in the yeast SSA assay as previously described. Targets are designed as pseudo-palindromic sequences (two identical recognition sequences are placed facing each other on both DNA strands). BurrH_36 based nucleases cleavage activity levels on their respective targets in yeast are shown in Table 14.
  • Table 14 Activity of BurrH_36 derived nuclease on one of the pseudo-palindromic sequences targets listed in Table 9 in yeast SSA assay at 37°C. + indicates a low activity, ++ a medium activity and +++ a high activity.
  • DNA target pCLS18476 cleavage activity pCLS18477 cleavage activity pCLS18478 cleavage activity pCLS18479 cleavage activity RAGT2.4 +++ + ++ ++
  • Example 6 Nuclease activity of engineered BurrH_36 based constructs in mammalian cells (CHO-K1) on an extrachromosomal target
  • DNA encoding burrH_36 based nuclease scaffold was extracted from yeast plasmid pCLS17028 using Ncol and BamHI.
  • the DNA insert was further ligated in NcoI/BamHI opened mammalian expression plasmids leading to BurrH_36 backbone plasmid pCLS18645 (SEQ ID NO. 232).
  • This mammalian expression plasmid encodes a Nuclear Localization Sequence (NLS) followed by HA tag, the BurrH_36 backbone and a Fokl catalytic head under an EF1a promoter.
  • NLS Nuclear Localization Sequence
  • burrH_36 based nuclease scaffold was extracted from yeast plasmid pCLS17028 (SEQ ID NO. 121) using EcoRV and BamHI.
  • the final BurrH_36 backbone plasmid pCLS18646 (SEQ ID NO. 233) was obtained by ligation of 3 fragments: the NcoI/BamHI opened mammalian expression plasmids, the EcoRV/BamHI digested burrH_36 based nuclease scaffold and a Ncol/EcoRV digested fragment encoding a Nuclear Localization Sequence (NLS) followed by S tag (SEQ ID NO. 234).
  • This mammalian expression plasmid encodes a Nuclear Localization Sequence (NLS) followed by S tag, the BurrH_36 backbone and a Fokl catalytic head under an EF1a promoter.
  • the DNA binding array inserts were then subcloned, from pCLS18123 and pCLS18127 (SEQ ID NO. 218 and SEQ ID NO.219) into pCLS18646 and pCLS18645 respectively (SEQ ID NO. 233 and SEQ ID NO. 232) using the two BsmBI sites, leading to respectively pCLS19041 (SEQ ID NO. 235) and pCLS19042 (SEQ ID NO. 236).
  • All the mammalian target reporter plasmids containing the DNA target sequences were constructed using standard gateway Gateway protocol (INVITROGEN) into a CHO reporter vector (Grizot, Epinat et al.; Arnould, Chames et al. 2006).
  • Activity of BurrH_36 based nucleases were tested in our extrachromosomal assay in mammalian cells (CHO K1) as homodimer (two identical recognition sequences are placed facing each other on both DNA strands) on the sequence target RAGT2.4 and RAGT2.3 (SEQ ID NO. 222 and SEQ ID NO. 223, Table 9).
  • CHO K1 cells were transfected in a 96-well plate format with 75 ng of target vector and an increasing quantity of each variant DNA from 0.02 to 25 ng, in the presence of PolyFect reagent (1 ⁇ L per well). The total amount of transfected DNA was completed to 100 ng (target DNA, variant DNA, carrier DNA) using an empty vector. 72 hours after transfection, culture medium was removed and 150 ⁇ l of lysis/revelation buffer for ⁇ -galactosidase liquid assay was added. After incubation at 37°C, OD was measured at 420 nm. The entire process is performed on an automated Velocity1 1 BioCel platform (Grizot, Epinat et al.).
  • Example 7a Nuclease activity of engineered BurrH_36 based constructs containing 18 DNA binding modules in mammalian cells (293H) at an endogenous locus
  • the DNA binding array inserts pCLS19087 and pCLS20851 (SEQ ID NO. 238 and SEQ ID NO. 239) were subcloned in mammalian expression plasmids pCLS18645 and pCLS18646 (SEQ ID NO. 232 and SEQ ID NO. 233) as described in example 6 leading to pCLS19638 and pCLS19679 respectively (SEQ ID NO. 240 and SEQ ID NO. 241). 293H cells were first plated at a density of 1.2 x 10 6 cells per 10 cm dish.
  • next day (day 0) cells were transfected with a total amount of 5 ⁇ g of pCLS19638 and pCLS19679 (SEQ ID NO. 240and SEQ ID NO. 241) or control empty vector using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's protocol.
  • Three days post-transfection (day 3) genomic DNA was extracted and the locus of interest was amplified with locus primers 1 and 2 (SEQ ID NO. 242 and SEQ ID NO. 243).
  • locus primers 1 and 2 SEQ ID NO. 242 and SEQ ID NO. 243
  • Primer 1 containing an adaptor sequences required for deep sequencing method using the GS Junior 454 (Roche). Examples of targeted mutagenesis (indels) at the desired locus (wt; SEQ ID NO. 237) are provided in Figure 10 .
  • the DNA binding array inserts pCLS20311 and pCLS20312 (SEQ ID NO. 245 and SEQ ID NO. 246) were subcloned in mammalian expression plasmids pCLS18645 and pCLS18646 (SEQ ID NO. 232 and SEQ ID NO. 233) as described in example 6 leading to pCLS21603 and pCLS21607 respectively (SEQ ID NO. 247 and SEQ ID NO. 248).
  • 293H cells were first plated at a density of 1.2 x 10 6 cells per 10 cm dish.
  • next day (day 0) cells were transfected with a total amount of 5 ⁇ g of pCLS21603 and pCLS21607 (SEQ ID NO. 247 and SEQ ID NO. 248) or control empty vector using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's protocol.
  • day 3 Three days post-transfection (day 3), genomic DNA was extracted and the locus of interest was amplified with locus primers 1 and 2 (SEQ ID NO. 242 and SEQ ID NO. 243).
  • locus primers 1 and 2 SEQ ID NO. 242 and SEQ ID NO. 243
  • Primer 1 containing an adaptor sequences required for deep sequencing method using the GS Junior 454 (Roche). Examples of targeted mutagenesis (indels) at the desired locus (wt; SEQ ID NO. 244) are provided in Figure 11 .
  • Example 7b Nuclease activity of engineered BurrH_36 based constructs containing 20 DNA binding modules in mammalian cells (293H) at an endogenous locus
  • the DNA binding array inserts pCLS20313 and pCLS20314 were subcloned in mammalian expression plasmids pCLS18645 and pCLS18646 (SEQ ID NO. 232 and SEQ ID NO. 233) as described in example 6 leading to pCLS21604 and pCLS21608 respectively (SEQ ID NO. 252 and SEQ ID NO. 253).
  • 293H cells were first plated at a density of 1.2 x 10 6 cells per 10 cm dish. The next day (day 0) cells were transfected with a total amount of 5 ⁇ g of pCLS21604 and pCLS21608 (SEQ ID NO. 252 and SEQ ID NO. 253) or control empty vector using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's protocol. Three days post-transfection (day 3), genomic DNA was extracted and the locus of interest was amplified with locus primers 1 and 2 (SEQ ID NO. 242 and SEQ ID NO. 243). Primer 1 containing an adaptor sequences required for deep sequencing method using the GS Junior 454 (Roche). Examples of targeted mutagenesis (indels) at the desired locus (wt; SEQ ID NO. 357) are provided in Figure 12 .
  • Example 8 Homologous Gene Insertion induced by engineered BurrH_36 based constructs (20 DNA binding modules) in mammalian cells (293H) at endogenous locus
  • the DNA binding array inserts pCLS20313 and pCLS20314 (SEQ ID NO. 250 and SEQ ID NO. 251) were subcloned in mammalian expression plasmids pCLS18645 and pCLS18646 (SEQ ID NO. 232 and SEQ ID NO. 233) as described in example 6 leading to pCLS21604 and pCLS21608 respectively (SEQ ID NO. 252 and SEQ ID NO. 253).
  • 293H cells were first plated at a density of 1.2 x 10 6 cells per 10 cm dish.
  • next day (day 0) cells were transfected with a total amount of 2.5 ⁇ g of pCLS21604 (SEQ ID NO. 252), 2.5 ⁇ g pCLS21608 (SEQ ID NO. 253), 5 ⁇ g of insertion matrix pCLS9893 (SEQ ID NO. 254), 250ng of GFP expression vector and completed to 15 ⁇ g with a control empty vector using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's protocol.
  • day 3 Three days post-transfection (day 3), cells were re-seeded in three 96 well plates at 10 cells per well and let at 37°C for 15 more days 5% CO 2 in DMEM complete medium.
  • TGI Targeted Gene Insertion
  • BurrH_36 (SEQ ID NO.2) was chosen as a starting polypeptide to create new specific nuclease.
  • Three hybrid C-terminal domains were constructed by the fusion of the C-terminal domain of BurrH36 to different fragments of C-terminal domain of AvrBs3 (SEQ ID NO. 259 to SEQ ID NO. 261).
  • the DNA coding for hybrid C-terminal domains (SEQ ID NO. 262 to 264) was synthesized according to the human genetic code and cloned in the pUC57 plasmid.
  • Inserts were obtained by standard molecular biology techniques (Pmll and BamHI restriction) and further subcloned in pCLS17419 (SEQ ID NO.124), leading to pCLS19785, pCLS19787 and pCLS19788 (SEQ ID NO. 265 to 267).
  • the DNA binding array insert was then subcloned from pCLS18120 (SEQ ID NO. 160) as described in example 3, into pCLS19785 and pCLS19787 to pCLS19788 leading to pCLS19815 to pCLS19817 (SEQ ID NO. 268 to 269). All molecular biology steps were done according to standard procedures.
  • BurrH_36 based nucleases were tested, as described in example 3, on the Avr15 target (SEQ ID NO. 192 shown in Table 6) at 37°C in yeast SSA assay as previously described ( WO 2004/067736 and (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006). BurrH_36 based nucleases cleavage activity levels in yeast are shown on Table 15. Table 15: Activity of BurrH_36 derived nuclease on AVR15 sequences targets in yeast SSA assay at 37°C. +++ indicates a high activity. DNA target pCLS19815 cleavage activity pCLS19816 cleavage activity pCLS19817 cleavage activity Avr15 +++ +++ ++++++
  • BurrH_36 (SEQ ID NO.2) was chosen as a starting polypeptide to create new specific nuclease.
  • Two hybrid C-terminal domains were constructed by the fusion of C-terminal domain of burrH36 to nuclear export signal sequence to regulate the quantity of burrH 36 in the nucleus (SEQ ID NO. 271 and SEQ ID NO. 272).
  • the DNA coding for hybrid C-terminal domains (SEQ ID NO. 273 and SEQ ID NO. 274) was synthesized according to the human genetic code and cloned in the pUC57 plasmid.
  • Inserts were obtained by standard molecular biology techniques (Xmal and BamHI restriction) and further subcloned in pCLS18645 and pCLS18646 (SEQ ID NO. 232 and SEQ ID NO. 233) leading to pCLS22405, pCLS22406, pCLS22420 and pCLS22421 respectively (SEQ ID NO. 275 to SEQ ID NO. 278).
  • a DNA binding array insert was then subcloned from pCLS19087 (SEQ ID NO. 238) as described in example 3, into pCLS22405 and pCLS22406 leading to pCLS23511 and pCLS23513 (SEQ ID NO. 280 to 281).
  • a DNA binding array insert was then subcloned from pCLS19088 (SEQ ID NO. 279) as described in example 3, into pCLS22420 and pCLS22421 leading to pCLS23531 and pCLS23533 (SEQ ID NO. 282 and SEQ ID NO. 283). All molecular biology steps were done according to standard procedures.
  • Nuclease pairs were tested in CHO as described in example 6 on the CAPT1 target (SEQ ID NO. 284), constructed as reported in example 6. Nuclease cleavage activity levels on their respective targets in mammalian cells are shown in Figure 14 .
  • Example 10 N-terminal fusion of the Fokl nuclease calalytic domain and activity of the N/N and C/N architectures
  • BurrH_36 nuclease described in the preceding examples comprise the catalytic domain Fokl fused to the C-terminal domain of the DNA binding domain.
  • the two others architectures of burrH_36 nuclease were evaluate for their ability to cleave DNA target.
  • BurrH_36 nuclease with N-terminal fusion of Fokl nuclease catalytic domain was engineered and activity of the N/N and C/N architectures was determined.
  • BurrH_36 (SEQ ID NO.2) was chosen as a starting polypeptide to create new specific burrH_36 nuclease with N-terminal fusion of Fokl nuclease catalytic domain.
  • the DNA coding for the Fokl catalytic domain, BurrH_36 N-terminal (SEQ ID NO.7) and hybrid C-terminal domains (SEQ ID NO. 262) was synthesized according to the human genetic code and cloned in the pUC57 plasmid, by Genecust (SEQ ID NO. 285).
  • NLS Nuclear Localization Sequence
  • yeast target reporter plasmid collections containing, either two AvrBs3 (SEQ ID NO. 290) sequence facing each other in the 5'/5' (or N/N) orientation on both DNA strand (SEQ ID NO. 291 to SEQ ID NO. 321, Table 16) or a single RAGT2.4 DNA target sequences (SEQ ID NO. 322) preceding a single AvrBs3 (SEQ ID NO. 290) target sequence on the same DNA strand (SEQ ID NO. 323 to SEQ ID NO. 358, Table 17) were constructed as previously described (International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al.
  • BurrH_36 based nucleases cleavage activity levels on the collection of targets in yeast are shown in Table 18.
  • Table 18 Activity of BurrH_36 derived nuclease on one of the pseudo-palindromic sequences targets listed in Table 9 and 17 in yeast SSA assay at 37°C.
  • Example 11 DNA targeting using polymorphism in position 13 while preceding position is constantly occupied by asparagines
  • DNA binding domain was synthesized according to the human genetic code and cloned in the pUC57 plasmid (SEQ ID NO. 359).
  • DNA binding domain was engineered to target the RAGT2.3 sequence (SEQ ID NO. 360) using in position 13 the following code: I to target A, N to target G, G to target T and D to target C. Insert was obtained by standard molecular biology techniques (BsmBI restriction) and further subcloned in pCLS17419 (SEQ ID NO.124), leading to pCLS21549 (SEQ ID NO. 361).
  • BurrH_36 based nucleases was tested on the RAGT2.3 and RAGT2.4 targets (SEQ ID NO.222 and SEQ ID NO 223) at 37°C in the yeast SSA assay as previously described. Targets are designed as pseudo-palindromic sequences (two identical recognition sequences are placed facing each other on both DNA strands). BurrH_36 based nucleases cleavage activity levels on the targets in yeast are shown in Table 19.
  • DNA binding domain was synthesized according to the human genetic code and cloned in the pUC57 plasmid (SEQ ID NO. 359).
  • DNA binding domain was engineered to target the RAGT2.4 sequence (SEQ ID NO. 322) using the following code in position 13: S to target A, N to target G, G to target T and D to target C. Insert was obtained by standard molecular biology techniques (BsmBI restriction) and further subcloned in pCLS20474 (SEQ ID NO. 288), leading to pCLS21558 (SEQ ID NO. 363).
  • BurrH_36 based nucleases was tested on the RAGT2.3 and RAGT2.4 targets (SEQ ID NO.222 and SEQ ID NO.223) at 37°C in the yeast SSA assay as previously described. Targets are designed as pseudo-palindromic sequences (two identical recognition sequences are placed facing each other on both DNA strands). BurrH_36 based nucleases cleavage activity levels on the targets in yeast are shown in Table 19.
  • DNA binding domain was synthesized according to the human genetic code and cloned in the pUC57 plasmid (SEQ ID NO. 364).
  • DNA binding domain was engineered to target the RAGT2.4 sequence (SEQ ID NO. 322) using the following code in position 13: I to target A, R to target G, G to target T and D to target C. Insert was obtained by standard molecular biology techniques (BsmBI restriction) and further subcloned in pCLS20474 (SEQ ID NO. 288), leading to pCLS21559 (SEQ ID NO. 365).
  • BurrH_36 based nucleases was tested on the RAGT2.3 and RAGT2.4 targets (SEQ ID NO.223 and SEQ ID NO.222) at 37°C in the yeast SSA assay as previously described. Targets are designed as pseudo-palindromic sequences (two identical recognition sequences are placed facing each other on both DNA strands). BurrH_36 based nucleases cleavage activity levels on the targets in yeast are shown in Table 19. Table 19: Activity of BurrH_36 derived nuclease on RAGT2.3 and RAGT2.4 sequences targets in yeast SSA assay at 37°C. - indicates no detectable activity, + indicates a low activity, and +++ a high activity. DNA Target pCLS21549 cleavage activity pCLS21558 cleavage activity pCLS21559 cleavage activity RAGT2.3 +++ + - RAGT2.4 - +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +
  • Example 12 Activity of hybrid nuclease (TALE-BurrH) created by replacing the N-terminal domain of BurrH by the N-terminal domain of AvrBs3
  • BurrH_36 (SEQ ID NO.2) was chosen as a starting polypeptide to create new specific nuclease. Based on 3D model of burrH_36 and PthXo1-derived TALE structure, point of mutations have been designed in order to minimize the possible steric hindrance between N-terminal domain of AvrBs3 and the first module of BurrH_36. The point mutations have been applied separately on the first module of BurrH36 (Architecture 1) or on the AvrBs3 N-terminal domain (Architecture 2).
  • Hybrid TALE-BurrH nuclease was constructed by the fusion of the truncated ⁇ 152 N-terminal domain of AvrBs3 (SEQ ID NO. 366) to the BurrH DNA targeting core.
  • Four amino acid residues of the first DNA binding module of BurrH were mutated (Q3P, V7A, A10T and A16T).
  • Example 13 Activity of BurrH-based nuclease containing mutations in the N-terminal domain
  • N-terminal domain of BurrH_36 (SEQ ID NO.2) was chosen as a starting polypeptide to create new N-terminal domains. Amino acid residues were mutated and/or inserted in the N-terminal domain of BurrH_36 to enhance the activity of the BurrH nuclease (see Figure 16 ).
  • the constructs containing an NLS a HA tag and various BurrH N-terminal domains containing mutations and or insertion were synthesized according to the human genetic code and cloned in the pUC57 plasmid leading to respectively pCLS20653 to pCLS20662 (SEQ ID NO. 379 to 388).
  • Inserts were obtained by standard molecular biology techniques (Ncol and Xmal restriction) and further subcloned in pCLS17419 (SEQ ID NO.124), leading to respectively pCLS21492 to pCLS21501 (SEQ ID NO. 389 to 398).
  • the DNA binding array inserts were then subcloned, pCLS18120 (SEQ ID NO. 160) into pCLS21492 to pCLS21501 using the two BsmBI sites (as described in Example 3), leading to respectively pCLS21512 to pCLS21521 (SEQ ID NO. 399 to 408). All molecular biology steps were done according to standard procedures.
  • BurrH_36 based nucleases were tested, as described in example 3, on the Avr15 target (SEQ ID NO. 192 shown in Table 6) at 37°C in yeast SSA assay as previously described ( WO 2004/067736 and (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006). BurrH_36 based nucleases cleavage activity levels in yeast are shown on Table 21. Table 21: Activity of BurrH_36 derived nuclease containing mutations in the N-terminal domain on Avr15 sequence target in yeast SSA assay at 37°C.
  • BurrH nuclease constructs Cleavage activity on Avr15 target sequence pCLS21512 +++ pCLS21513 +++ pCLS21514 +++ pCLS21515 +++ pCLS21516 +++ pCLS21517 +++ pCLS21518 +++ pCLS21519 +++ pCLS21520 +++ pCLS21521 +++
  • Example 14 BurrH-based sequence-specific nucleases create mutations at an endogenous locus in plants
  • a BurrH-based nuclease (SEQ ID NO: 409 and SEQ ID NO: 410) was engineered to recognize a site within the first 90 bp of the coding sequence of the ⁇ 1,2-xylosyltransferase (XylT1) gene of Nicotiana benthamiana.
  • XylT1 ⁇ 1,2-xylosyltransferase
  • N. benthamiana seeds were surface sterilized and plated on agarose medium containing Murashige and Skoog salts with an iron supplement.
  • Protoplasts were isolated from young expanded leaves using the protocol described by Wright et al, 2005. Plasmids were introduced separately into aliquots of protoplasts through PEG-mediated transformation (Yoo et al 2007).
  • YFP YFP(SEQ ID NO: 411)
  • SEQ ID NO: 409 and SEQ ID NO: 410 Another encodes the BurrH-based nuclease (SEQ ID NO: 409 and SEQ ID NO: 410).
  • Protoplasts were transformed with 12 ⁇ g of each plasmid. Twenty-four hours after transformation, the protoplasts that had been transformed with the YFP-encoding plasmid were subjected to fluorescence microscopy to assess transformation efficiency. More than 90% of the protoplasts expressed YFP. Genomic DNA was then prepared from each of the three aliquots of transformed protoplasts.
  • I-Tevl The catalytic domain of I-Tevl (SEQ ID NO. 413), a member of the GIY-YIG endonuclease family was fused to BurrH-36 backbone derived from E5AV36 BURRH protein.
  • I-Tevl is a tripartite protein composed of a C-terminal domain responsible for binding specificity, linked to N-terminal catalytic domain by a long flexible linker.
  • the N-terminal catalytic domain contributes to specificity via DNA cleavage selectivity, characterized biochemically and defined by the degenerate CNNNG motif (with CAACGC as the natural cleavage sequence).
  • the BurrH_36 core scaffold (SEQ ID NO. 414) derived from E5AV36 BURRH protein was composed of a N-terminal domain and a C-terminal domain separated by a small DNA sequence containing two BsmBI sites allowing further cloning of the nucleic acid sequence coding for the DNA binding array. Short sequences were added between the different pieces for cloning purpose or to create linkers at the protein level.
  • the BurrH_36 scaffold was then cloned into vector pCLS7865 (SEQ ID NO. 415) to generate pCLS7865-BurrH_36.
  • TevD02 SEQ ID NO. 416
  • TevM01 SEQ ID NO. 417
  • TevD02 and TevM01 were fused to the N-terminal part of the BurrH_36 scaffold into the pCLS7865-BurrH_36 by restriction and ligation using standard biological tools, yielding pCLS7865-TevD02::b36 and pCLS7865-TevM01::b36.
  • the TevD02::b36 fusion contains the sequence -QGPSG- linking the BurrH_36-derived DNA binding domain and TevD02 catalytic domain and the TevM01::b36 fusion contains the dipeptide -IA- linking the BurrH_36-derived DNA binding domain and TevM01 catalytic domain.
  • the nucleic acid sequence coding for the DNA binding array to target the AvrBs3 site was subcloned into the plasmid pCLS7865-TevD02::b36 and pCLS7865-TevM01::b36 by restriction and ligation using standard biological tools to create the subsequent TevD02::b36-AvrBs3 and TevM01::b36-AvrBs3 constructs respectively.
  • TevD02::b36-AvrBs3 and TevM01::b36-AvrBs3 yeast expression plasmids encoding TevD02-burrH and TevM01-burrH chimeric endonucleases (SEQ ID NO.421 and SEQ ID NO. 422) were prepared by yeast in vivo cloning using TevD02::b36-AvrBs3 and TevM01::b36-AvrBs3 constructs.
  • TevD02::b36-AvrBs3 or TevM01::b36-AvrBs3 plasmid linearized were used to transform the yeast S . cerevisiae strain FYC2-6A ( MAT ⁇ , trp1 ⁇ 63, leu2 ⁇ 1, his3 ⁇ 200 ) using a high efficiency LiAc transformation protocol (Arnould, 2007).
  • yeast target reporter plasmids containing the MBBBD DNA target sequence were constructed as previously described ( WO 2004/067736 ; Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).
  • TevD02::b36-AvrBs3 and TevM01::b36-AvrBs3 constructs were tested in a yeast SSA Assay as previously described on the DNA target containing the terminal G base of I-Tevl cleavage sequence CAACGC (SEQ ID NO.424) spaced away of 10 bp from the residue preceded the AvrBs3 recognition site (SEQ ID NO. 425) (CAACGC-10N-AvrBs3 recognition site).
  • TevD02::b36-AvrBs3 and TevM01::b36-AvrBs3 activity levels on the DNA target sequence in yeast cell are shown inTable 22.
  • Table 22 Activity of monomeric MBBBD nuclease in yeast (37°C). Activity of TevD02::b36-AvrBs3 and TevM01::b36-AvrBs3 on DNA target containing natural I-Tevl cleavage site (CAAGC) wherein the terminal G base of the I-Tevl cleavage site is spaced away of 10 bp from the residue preceded the single AvrBs3 recognition site (SEQ ID NO. 425).
  • the negative control consists in a TALE scaffold without any DNA binding domain. n.d. indicates no detectable activity, +++ indicates an activity over 0.7 in yeast SSA assay. Target sequence Neg. Control TevD02::b36-AvrBs3 TevM01::b36-AvrBs3 CAAAGCNNNNNNNNNNATAAACCTAACCCTCT n.d. +++ +++
  • Example 16 Activity of Tevl::b36 and Tevl::cT11 in mammalian cells (CHO-K1) on a chromosomal target
  • Tevl::b36 based nucleases to induce targeted mutagenesis on a chromosomal target was monitored using an engineered cell line (CHOpi-10, ref: patent US20120272348 A1 ) having a single integrated copy of a GFP-encoding sequence under the control of a CMV promoter.
  • a DNA binding array was synthesized (RVD_bhEGFP_T03g06; SEQ ID NO. 426) to target a unique sequence within the encoded GFP gene (SEQ ID NO. 427), thus allowing for measuring in vivo mutagenic activity using two complimentary methods: (i) via a reduction in GFP-positive cells as determined by flow cytometry, and; (ii) via direct amplicon sequencing of the targeted region.
  • the core TevM01::b36 scaffold insert was first transferred from pCLS7865-TevM01::b36 to pCLS1853 (SEQ ID NO. 428) to create plasmid pCLS21536.
  • the DNA binding array insert from RVD_bhEGFP_T03g06 was subcloned into pCLS21536, yielding plasmid pCLS20293.
  • the final pCLS20293 vector contains the coding sequence for the Tevl::b36EGfpT3g6 construct (SEQ ID NO. 429) which targets GFP and whose expression is controlled by a CMV promoter.
  • the sT2 (SEQ ID NO: 433) core TALE scaffold was selected to generate pCLS7865-cTAL11_NFS1 (pCLS9008, SEQ ID NO: 434), where NFS1 designates the amino acid sequence -GSSG- (with underlying restriction sites BamHI and Kpn2l in the coding DNA to facilitate cloning).
  • TevM01 was subcloned into the pCLS9008 backbone, yielding pCLS7865-TevM01::cT11.
  • the fusion contains the sequence -SG- linking the TALE-derived DNA binding domain and I-Tevl-derived catalytic domain.
  • Tevl::cT11 based nucleases to induce targeted mutagenesis on a chromosomal target was monitored using an engineered cell line (CHOpi-10, ref: patent US20120272348 A1 ) having a single integrated copy of a GFP-encoding sequence under the control of a CMV promoter.
  • a DNA binding array was synthesized (RVD_ctEGFP_T03g12-L1; SEQ ID NO.
  • the core TevM01 ::cT11 scaffold insert was first transferred from pCLS7865-TevM01::cT11 to pCLS1853 (SEQ ID NO. 428) to create plasmid pCLS20650 (SEQ ID NO. 437).
  • the DNA binding array insert from RVD_ctEGFP_T03g12-L1 was subcloned into pCLS20650, yielding plasmid pCLS20790.
  • the final pCLS20790 vector contains the coding sequence for the Te-vl::cT11EGfpT3g12 construct (SEQ ID NO. 438), which targets GFP and whose expression is controlled by a CMV promoter.
  • BurrH_36 Effector scaffold was engineered. This scaffold consisted in a regular Burrh_36 scaffold (pCLS23330, SEQ ID NO. 439) fused via its C terminal end, to the VP64 transcription activator domain. To allow quantitative assessment of transfection efficiency of Burrh_36 Effector plasmid, a self cleaving Green Fluorescent Protein (GFP) was fused to the C terminal domain of the VP64 via a 2A self-cleavage peptide. This construction, named pCLS23453 (SEQ ID NO. 440), was used as recipient backbone for the subcloning of two different Burrh_36 DNA binding arrays.
  • GFP Green Fluorescent Protein
  • the BurrH_36 WT and BurrH_36 HBB DNA binding arrays containing respectively 20 and 16 DNA binding modules and targeting respectively the DNA binding sites SEQ ID NO. 443 and SEQ ID NO. 444, were subcloned in pCLS23453 using BsmBI insertion sites.
  • the activities of the resulting Burrh_36 effectors (pCLS23638 and pCLS23636; respectively SEQ ID NO. 441 and SEQ ID NO. 442) were then assayed using a transcription activation reporter plasmid.
  • This ectopic reporter plasmid contained one of the DNA binding sites mentioned above (SEQ ID NO. 443 and SEQ ID NO.
  • the reporter plasmid located upstream from a minimal cytomegalovirus (CMV) promoter driving the Blue Fluorescent Protein (BFP, SEQ ID NO. 449) reporter gene.
  • CMV minimal cytomegalovirus
  • BFP Blue Fluorescent Protein
  • the reporter plasmid also contained a Red Fluorescent Protein (DsRed, SEQ ID NO. 450) constitutively driven by a chimeric promoter encompassing a SV40 early promoter fused to an EM7 promoter.
  • DsRed Red Fluorescent Protein
  • Burrh_36 Effector-dependent induction of BFP signal was determined in 293H cells by flow cytometry, according to the procedure described in the following. Briefly, 293H cells were transfected in 10 cm plate format (1.2 10 6 cells/well), using 3000 ng of reporter plasmid and increasing amount of Effector plasmid (0, 500 or 1000 ng), using Lipofectamine as a transfection agent (25 ⁇ L/plate). For each experiment, the total DNA content was adjusted 4000 ng/plate with an empty vector (pCLS0003, SEQ ID NO 430).
  • Table 25 Sequence identity matrix showing percentages of identity between the N-terminal amino acids sequences of E5AV36, E5AW45 and E5AW46 and AvrBs3. Seq-> N-ter AvrBs3 E5AV36 E5AW45 E5AW43 AvrBs3 ID 0.065 0.061 0.079 E5AV36_Nter 0.065 ID 0.752 0.576 E5AW45_Nter 0.061 0.752 ID 0.626 E5AW43_Nter 0.079 0.576 0.626 ID Table 26: Sequence identity matrix showing percentages of identity between the C-terminal amino acids sequences of E5AV36, E5AW45 and E5AW46 and AvrBs3.
  • N-ter Modules: LEPKD IVSIA SNNGA TQAIA TLLAK WDSLI AKG (SEQ ID NO.77) LQPKD IVSIA SHGGA TQAIT TLLNR WGDLR AKE (SEQ ID NO.78) LEPKD IVSIA SHDGA TQAIT TLLEK WDELR AKG (SEQ ID NO.79) LEPKD IVSIA SHIGA NQTIT TLLNK WGALI DLE (SEQ ID NO.80) LEPKD IVSIA SHGGA NKAIT TLLEK WAALR AKE (SEQ ID NO.81) LEPKD IVSIA SHNGA TQAIT TLLEK WGDLR AKE (SEQ ID NO.82) LEPKD IVSIA SNTGA NKTIT RLLEK WGDLR AKE (SEQ ID NO.83) LEPKD IVSIA SHDGS NQTIT KLLEK WDELR AKG (SEQ ID NO.84) LEPKD IVSIA SHIGA NQTIT TLLNK WGALI DLE
  • N-ter WSNKAITTLLENWEKLIKKG (SEQ ID NO.76) Modules: LKPED IVTIA SHHGG SQAIT TLLEN WDDLL KLE (SEQ ID NO.106) LKFED IVSIA SHNGA SQAIT TLLEN WEKLI KKG (SEQ ID NO.107) LKPED IVSIA SHSGG SQAIT TLLEN WDDLI DQE (SEQ ID NO.108) C-ter: YTESE IVNIF SSQDG VLKLL AELD (SEQ ID NO.109) Table 34: List of peptide linkers that can be used in MBBBD proteins.

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Claims (9)

  1. Polypeptide qui comprend une succession de modules de 30 à 35 acides aminés, ladite succession de modules présentant une spécificité base par base envers une séquence cible d'acide nucléique, dans lequel
    (i) au moins un desdits modules présente au moins 70 % d'identité de séquence avec une des séquences des modules polypeptidiques de la protéine E5AV36 de SEQ ID NO: 2 de Burkholderia rhizoxinica et présente au moins 90 % d'identité de séquence avec une séquence polypeptidique sélectionnée dans le groupe consistant en : SEQ ID NO: 162 à 181,
    (ii) ledit au moins un des modules comprend, à la position 13, un résidu d'acide aminé variable unique qui détermine la spécificité de chaque module envers une base nucléotidique de la manière suivante : AA Base nucléotidique I, S, T A G, R T D, T, * C N, R G
    où « * » signifie une absence d'acide aminé, et
    (iii) ladite succession de modules est fusionnée à un domaine catalytique possédant une activité endonucléase sous une forme monomérique ou dimérique.
  2. Polypeptide selon la revendication 1, dans lequel la séquence d'acides aminés dudit au moins un module est sélectionnée dans le groupe consistant en SEQ ID NO: 11 à 30.
  3. Polypeptide selon la revendication 1 ou la revendication 2, dans lequel ladite séquence d'acide nucléique cible ne comprend pas de nucléotide thymidine à la position -1.
  4. Polypeptide selon l'une quelconque des revendications 1 à 3, où ledit polypeptide comprend en outre un module qui présente au moins 80 % d'identité d'acides aminés avec une répétition AvrBs3 de SEQ ID NO: 10.
  5. Polypeptide selon l'une quelconque des revendications 1 à 4, dans lequel ledit domaine catalytique provient de l'endonucléase FokI ou d'une endonucléase de ciblage (« homing »).
  6. Polypeptide selon l'une quelconque des revendications 1 à 4, dans lequel ledit domaine catalytique provient de I-TevI.
  7. Utilisation d'un polypeptide selon l'une quelconque des revendications 1 à 6, pour la transformation in vitro du matériel génétique d'une cellule au sein de ou en position adjacente à ladite séquence cible d'acide nucléique.
  8. Polypeptide selon l'une quelconque des revendications 1 à 6, destiné à être utilisé en tant que médicament.
  9. Procédé in vitro pour cibler le matériel génétique d'une cellule, comprenant :
    (a) la mise à disposition d'une cellule, de préférence une cellule de mammifère ou une cellule végétale comprenant une séquence cible d'acide nucléique ; et
    (b) l'introduction dans la cellule d'un polynucléotide selon l'une quelconque des revendications 1 à 8, et facultativement d'un acide nucléique exogène comprenant une séquence homologue à au moins une partie de la séquence cible d'acide nucléique, de sorte qu'une recombinaison homologue se produise entre ledit acide nucléique exogène et la séquence cible d'acide nucléique ;
    dans lequel le polypeptide codé par ledit polynucléotide transforme le matériel génétique de la cellule au sein de ou en position adjacente à ladite séquence cible d'acide nucléique.
EP13776595.4A 2012-07-24 2013-07-24 Nouveaux domaines de liaison d'acide nucléique spécifiques à la base modulaires à partir de protéines burkholderia rhizoxinica Active EP2877488B1 (fr)

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WO2014018601A2 (fr) 2014-01-30
WO2014018601A3 (fr) 2014-06-26
US10472396B2 (en) 2019-11-12
US20150225465A1 (en) 2015-08-13
EP2877488A2 (fr) 2015-06-03

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